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Muscadine grapes (Vitis rotundifolia) are native to the southeastern U.S., where they are valued for their unique flavor and fruity aroma. Despite having a diverse aroma profile, muscadine germplasm is virtually unexplored in terms of its aroma volatile content and composition, which is crucial in determining the value of its products. The aim of this research was to characterize 24 muscadine genotypes with distinct uses and origin for their aroma-related volatile profiles using the headspace solid-phase microextraction method coupled with gas-chromatography mass spectrometry. In total, 63 volatile compounds were detected, and genotypes significantly differed for 43 of the volatile compounds. We also profiled the aroma volatile content and composition of the commercially cultivated muscadine cultivar Carlos at various stages of berry ripeness. Characteristic differences were observed in the composition of the volatile compounds as ripening progressed. This is the first study to have evaluated the aroma volatile composition of a wide variety of muscadine germplasms, including juice and fresh fruit cultivars, as well as the related species Vitis popenoei and its complex hybrids between V. rotundifolia and Vitis vinifera. The results obtained from this study will help identify muscadine genotypes and better design crosses to produce fresh fruit and wine selections with the desired aroma profiles. This knowledge will lead to the development of new muscadine cultivars and significantly contribute to the expansion of muscadine use in the future.